ABSTRACT In over 50% of cases, activating mutations in the serine/threonine kinase BRAF are found to drive melanoma, the deadliest form of skin cancer. Canonically, oncogenic BRAF has been shown to promote unrestrained cellular proliferation by hyper-activating the MAPK signaling pathway. However, whether oncogenic BRAF promotes additional cellular defects that facilitate tumorigenesis remains relatively unexplored. Utilizing high- resolution fixed and live-cell imaging, coupled with in vivo zebrafish models, we have observed that expression of oncogenic BRAF severely impairs chromosome segregation, promotes cytokinesis failure and disrupts nuclear morphology. The primary objective of this proposal is to identify the underlying mechanisms by which oncogenic BRAF promotes these cellular defects. In Aim 1, we will define the mitotic defects imparted by oncogenic BRAF that disrupt normal chromosome segregation. In addition, we will use SILAC mass spectrometry to test the novel hypothesis that indiscriminant phosphorylation of key mitotic regulators by oncogenic BRAF underlies the generation of these observed mitotic defects. In Aim 2, we will test the hypothesis that oncogenic BRAF disrupts normal actin cytoskeletal contractility and/or inappropriately phosphorylates the nuclear lamina, thus promoting nuclear envelope rupture and the acquisition of DNA damage. In Aim 3, we will test the hypothesis that the Hippo tumor suppressor pathway, which limits the proliferation of spontaneously arising tetraploid cells, becomes activated secondary to oncogenic BRAF- induced cytokinesis failure. Hippo pathway activation will be explored both in vitro, following doxycycline- inducible expression of oncogenic BRAF in primary cell lines, and in vivo, in human and mouse primary tissue sections of benign nevi generated by oncogenic BRAF expression. We will also test the hypothesis that Hippo pathway inactivation restores proliferative ability to BRAF-generated tetraploid cells and thus may facilitate tumorigenesis. Collectively, these experiments will define novel roles of oncogenic BRAF in driving genome instability and promoting melanomagenesis.